Lactate is an organic chemical compound that participates in the metabolism of eukaryotic and prokaryotic cells. Lactate is exchanged between organelles, cells and organs as fuel or waste product, and also plays important signaling and biosynthetic roles, being involved in the physiology of exercise, inflammation, wound healing, neurovascular coupling and also in diseases such as cancer, hypoxic/ischemic disease and microbial infection. In addition, lactate is of industrial interest as a food additive, as a detergent, for the detection and control of microbial growth and for the production of biodegradable polymers.
Lactate is in dynamic flux between subcellular compartments, between the cell and the extracellular space and between cells. Because the concentration of lactate in the cell compartments is unknown, the dynamics of lactate in the living body is a largely unknown area.
Standard methods to measure lactate are based on enzymatic reactions, which have to be followed by photometric, amperometric or other devices. Enzyme-based electrodes have been developed that can detect lactate with high-temporal resolution. Another approach to measure lactate is high performance liquid chromatography (HPLC), where lactate is separated from other compounds by passing the sample through a stationary phase stored in a column. There is a problem in the prior art, however, that the existing methods are invasive as they require the extraction of samples or consume lactate, and therefore, they change the concentration of lactate in the sample. A second problem is their sensitivity, since they can not detect the minute amount of lactate present in a single cell or a single subcellular organelle.
The transport of lactate across cellular and subcellular membranes is mediated by the monocarboxylate transporter (MCT), a molecule involved in the pathogenesis of several diseases and an important target for pharmacological intervention in cancer and diabetes. There are no available methods to measure the transport of lactate in single cells. More specifically, current and common techniques used to measure the transport of lactate using radioactive isotopes cannot resolve single cells and have poor temporal resolution, which hampers the study of fast phenomena and normal tissues, which are heterogeneous in their cellular composition. An existing technique infers the transport of lactate in single cells from changes in pH that accompany the transport of lactate, but this technique is limited insofar as requires prior knowledge of the usually unknown buffering capacity of the cell and is not easily applicable in the presence of physiological bicarbonate buffers.
The rates of lactate production and lactate consumption are important parameters of cell metabolism, with relevance for hypoxia/ischemia, cancer, diabetes and other pathological conditions. There are no available methods to measure the rates of lactate production and consumption in single cells. More specifically, current and common techniques used to measure the rates of lactate production and consumption are enzyme-based methods that cannot resolve single cells, have poor temporal resolution, and cannot be applied in the presence of physiological concentrations of lactate. Particularly, measurements using isotopes cannot resolve single cells and have poor sensitivity and temporal resolution. Other currently available technique infers the production of lactate by a cell population by following changes in pH that accompany the production of lactate, but this indirect technique is limited insofar as it is affected by other mechanisms affecting extracellular pH and is not easily applicable in the presence of physiological bicarbonate buffers.
The rate of pyruvate consumption by mitochondria, equivalent under some conditions to the rate of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, is one of the fundamental parameters of cell metabolism and is affected in several diseases including hypoxic/ischemia, cancer, diabetes and other conditions. There are no available methods to measure the rates of the mitochondrial metabolism in single cells. More specifically, current and common techniques to measure the rates of the mitochondrial metabolism use cannot resolve single cells and have poor sensitivity and low temporal resolution.
In the state of the art there is no evidence of an optical tool or nanosensor for detecting and quantifying lactate in samples, in tissues and in cellular and subcellular compartments, with high spatial and temporal resolution. Also, there are no available techniques to quantitate single-cell resolution lactate transport or the rates of lactate consumption/production or the rate of mitochondrial metabolism or the Warburg effect, the metabolic transformation that underlies cancer. Nevertheless there are related documents in the art, which will be described below. Sensors for different metabolites are described in WO2006096213A1, WO2006096214A1, WO2006044612A2 and WO2007046786A2 that involve a FRET donor, a FRET acceptor and a member of the class of periplasmic binding proteins (PBPs), proteins located in outside bacterial plasma membranes involved in chemotaxis. The periplasmic binding protein serves as the specific recognition element. As there is no known rule to predict whether a given protein may serve as an effective recognition element, these proteins have been the result of informed trial and error, semi-rational design. The current invention does not used any of the recognition elements described WO2006096213A1, WO2006096214A1, WO2006044612A2 or WO2007046786A2. Moreover, the current invention is not based on any members of the periplasmic binding protein family but rather on a member of the GntR family, a subclass of transcription factors involved in adaptation of bacteria to changing environmental conditions. Surprisingly, the sensor described in the present invention was found to detect its ligand over 4 orders of magnitude, which makes it unique. PBP-based sensors can only quantify ligands over 2 orders of magnitude only.
WO2001033199A2 discloses a probe based on a target binding site peptide (i) attached to a first fluorescent polypeptide capable of binding to (i) and attached to a second fluorescent polypeptide. The probe includes a linker connecting the two fluorescent polypeptides which allows the distance between them to vary, the fluorescent polypeptides display fluorescence resonance energy transfer (FRET) between them. The probe described in WO2001033199A2 is qualitatively different from the probe described in the current invention insofar as the current invention does not involve displacement of binding between two peptides but rather a conformational change elicited by the ligand in a whole protein.
WO2008008149 describes a method to measure the rates of glycolysis and mitochondrial metabolism in cell populations by recording the rate of extracellular oxygen depletion and the rate of extracellular acidification over minutes using a specific dedicated apparatus. The current invention differs from WO2008008149 as it does not need a dedicated apparatus and can be used with standard multi-well plate readers. It also differs in terms of spatial resolution as it can measure single cells and temporal resolution, which is in the order of seconds. The current invention measures the rate of lactate production directly, whereas WO2008008149 provides an indirect estimate by recording the accumulation of extracellular protons, a parameter that is affected by other processes unrelated to metabolism and that required unphysiological pH buffering conditions.
WO/2012/002963 describes a method to estimate the rate of glucose consumption in single cells or cell population with high temporal resolution using a FRET glucose nanosensor. The current invention differs from WO/2012/002963 as is does not measure glucose or the rate of glucose consumption but the rates of lactate production/consumption and the rate of mitochondrial metabolism, rates that are independent of the rate of glucose consumption, being a completely different technical application. Moreover, the present method allows an estimation of the Warburg effect, which is not possible with a glucose nanosensor.